The primary goal of this research is to develop a highly sensitive and specific assay for the measurement of 2 important metabolites of vitamin D, 25-hydroxy- vitamin D and 1,25 dihydroxy vitamin D, in a non-radioactive assay. Preliminary work has demonstrated that a recently synthesized biotin- conjugate of 25-OH-D3 can be useful for measuring the concentration of 25-OH-D3 in a laboratory sample. A biotin conjugate of 1,25-(OH)2-D3 has also been synthesized and used for binding studies. The Phase I goal will be to use these recently synthesized reagents to develop a non-radioactive assay for quantification of 25-OH-D and 1,25-(OH)2-D3 in blood. This method could then be adopted other laboratories. Most of the reagents required have been synthesized, and the investigator has responded well to most of the previous concerns. However, critical controls and details are lacking in the experiments proposed. Thus, although this revised application is superior than the previous one, concern still exists and the proposal generates only moderate enthusiasm.